Stem cells have traditionally served as a valuable resource for models of development and morphogenesis, but they also factor significantly in the development of regenerative therapies aiming to treat various degenerative diseases and traumatic injuries. Pluripotent embryonic stem cells (ESCs) are capable of differentiating into an array of cell types, including functional neurons, cardiomyocytes and pancreatic beta cells, thus representing a robust cell source for biological studies and regenerative cell therapies. Despite the clear potential of pluripotent stem cells, a critical limitation is the inability to efficiently differentiate ESCs to specific cell fates in a reproducible, reliable and homogeneous manner. During embryonic development, the spatiotemporal fidelity of differentiation is precisely regulated by the local amount of morphogenic factors presented at the appropriate time with a finite duration. However, most in vitro methods typically used to examine the differentiation of stem cells in response to morphogen treatment lack the ability to uniformly present molecules in defined amounts with precise temporal resolution. Forced convection intercellular perfusion (FCIP) created by microfluidic culture systems can overcome the limitations of diffusive transport by presenting molecules to 3D cultures of cells in a uniform temporal and dose-dependent controlled manner. The inherent flexibility and scalability of microfluidic perfusion culture systems represents an innovative and translatable approach to enhance directed stem cell differentiation. Based on this rationale and significant preliminary data, the objectives of this proposal are to 1) define the transport limitations of 3D aggregates of differentiating ESCs, 2) determine the effects of perfusion culture on the yield and homogeneity of ESC differentiation, and 3) examine the dose and temporal effects of morphogen presentation on ESC differentiation in a high-throughput manner. The completion of these studies will yield novel information about the ability to more controllably direct the differentiation of ESCs by engineering the dynamic molecular composition of the extracellular microenvironment using microfluidic perfusion culture. The proposed approach represents a fundamentally new route to more efficiently direct the differentiation of stem cells in vitro through the simultaneous control of dose and temporal presentation of molecular factors locally, which may be a broadly applicable principle in the development of stem cell technologies. PUBLIC HEALTH RELEVANCE: The development of stem cell regenerative and diagnostic technologies is currently limited by an inability to efficiently control the differentiation of the stem cells. This proposal seeks to improve the efficiency and homogeneity of pluripotent stem cell differentiation by controlling the dose and timing of morphogenic factor presentation within 3D stem cell microenvironments via microperfusion culture systems.